To inhibit the devastating progression of autoimmune disease mediated by T lymphocytes, there is a critical need to block the function of the signal transducer and activator of transcription 3 (STAT3). STAT3 is required for lineage commitment of T helper 17 (Th17) cells, and Th17 cells are causally linked to the damage that affects target organs in complex autoimmune diseases such as multiple sclerosis, inflammatory bowel disease, psoriasis, and rheumatoid arthritis. The central hypothesis of this proposal is that inhibition of STAT3 will block the differentiation and destructive effects of autoimmune Th17 cells. The long-term goal is to identify small molecule inhibitors of STAT3 nuclear import with sufficient potency and selectivity that can lead to the development of drugs that will have a major impact in the treatment of autoimmune diseases. This proposal is in response to a Program Announcement to Develop Assays for High-Throughput Screening for use in Probe and Pre-Therapeutic Discovery. The objective is to develop an automated high content screening (HCS) imaging assay able to quantify nuclear STAT3 and to use this assay to screen small molecule libraries for compounds that inhibit STAT3 nuclear import. Identification of cell permeable molecules that specifically block the nuclear import of unphosphorylated and tyrosine phosphorylated STAT3 is expected to impact Th17 cell function in autoimmunity. A cell-based approach will be used to discover inhibitors of STAT3 nuclear import with three specific aims: 1) Develop and optimize a fluorescence-based microscopic assay for STAT3-GFP nuclear import on automated HCS imaging platforms. 2) Adapt and validate the performance of the STAT3-GFP nuclear import HCS assay in pilot compound library screens. 3) Create a testing paradigm of established secondary and tertiary assays to characterize compounds for selectivity, mechanism of action, and biological effects. The complexities of autoimmune pathologies make it unlikely that one therapy will work for every disease. The most effective and least toxic approaches are expected to target specific proteins or signaling pathways. STAT3 is one of the factors causally linked to autoimmunity, and blocking the action of STAT3 is expected to have a significant impact on clinical intervention. Identifying cell permeable inhibitors of STAT3 action will support the rational design of therapeutics.